The immune response of the mammalian immune system is generally divided into two general types: humoral immunity, mediated largely by circulating antibodies, and cellular immunity mediated by various forms of T-cells. Generally, extracellular antigens stimulate a humoral response, while intracellular antigens such as viruses, stimulate a cellular response.
The cellular immune response to virally infected cells and tumor cells is largely mediated by cytotoxic T-lymphocytes (T.sub.c, or CTL), when they recognize foreign antigens attached to the host cell surface as part of the Major Histocompatibility Complex (MHC), and more particularly, a common form of MHC known as MHC Class I. In contrast, antigens derived from non-viral pathogens (bacteria, fungi) are generally expressed as part of an MHC Class II complex. A different subpopulation of effector T cells (cell mediated immune cells; CMI) release cytokines that activate the host cell to destroy such pathogens.
In experimental systems, tumor-antigen specific CTL are the most powerful immunological mechanism for the elimination of tumors. CTL can be induced either in vivo with vaccines or can be generated in vitro and then be re-infused into the tumor-bearing organism. The in vivo induction of CTL is typically accomplished by immunization with live virus or cells (Tanaka, et al., J. Immunol. 147:3646-52 (1991); Wang, et al., J. Immunol. 4685-4692(1995); Torre-Amione, et al., Proc. Natl. Acad. Sci. U.S.A. 87:1486-90 (1990)).
Except for a few special viral proteins such as the SV-40 large T-antigen and the Hepatitis B surface antigen, injection of isolated or soluble proteins does not result in induction of CTL (Schirmbeck, et al., Eur. J. Immunol. 23:1528-34 (1993)). CTL are induced when a protein enters the class I pathway of antigen processing. To enter this pathway the protein must be present in the cytosol of the APC. There it is degraded into peptides which are then transported into the endoplasmic reticulum, where they associate with nascent HLA class I molecules. These peptides are then displayed together with the class I molecules on the cell surface and can serve as an inducer and target of class I restricted antigen-specific CTL. Physiologically, only proteins that are endogenously synthesized by an APC enter this pathway. Non-cellular-delivery vehicles for proteins, such as Ph-sensitive liposomes, can overcome the requirement for endogenous synthesis in vivo (Nair, et al., J. Exp. Med. 175:609-12 (1992); Nair, et al., J. Virol. 67:4062-9 (1993)); however, these treatments are also quite toxic to the target cells.
Induction of primary HLA class I restricted CTL by pure soluble proteins in vitro has not been reported. The most common tool for ex vivo induction of primary CTL are small (8-11-mer) synthetic peptides (Stauss, et al., Proc. Natl. Acad. Sci. U.S.A. 89:7871-5 (1992); Carbone, et al., J. Exp. Med. 167:1767-79 (1988)). These synthetic peptides associate with class I molecules on the cell surface without the requirement for endogenous processing. When presented on the surface of an appropriate APC (such as a dendritic cell) they can then induce a primary CTL response. However, frequently these CTL do not protect against challenge with pathogens that endogenously synthesize the protein from which the peptide was derived because of their low T-cell receptor avidity (Speiser, et al., J. Immunol. 149:972-80 (1992)) and because they induce reactivity with a single epitope of the target antigen.
GM-CSF is a cytokine that has pleiotropic function both in hematopoiesis as well as in immunology. GM-CSF has been shown to promote differentiation and survival of dendritic cells. GM-CSF can be used as an systemic adjuvant (Jones, et al., Eur. J. Clin. Microbiol. Infect. Dis. 13:S47-53 (1994)).
It is well known that immunization with soluble proteins can result in a significant antibody response. However, since class II restricted antigen presentation or direct B-cell stimulation is responsible for this effect, antibody induction has no predictive value for the induction of class I mediated induction of CTL. Most proteins that induce antibodies in vivo fail to induce CTL.
GM-CSF fusion proteins have been shown to induce in vivo antibody responses in a lymphoma mouse model (Levy and Tao, Nature 362:755-758 (1993); Chen, et al., J. Immunol. 3105-3117 (1995)). In this study, tumor idiotype fused to GM-CSF was found to be superior to the mixture of both molecules and to other conventional adjuvants for the induction of antibody responses. Contrary to other solid tumors antibodies are believed to be the effector mechanism for tumor protection and for tumor therapy in lymphoma.
Moreover, in vitro induction of immunity is generally much more difficult to achieve for both cellular and humoral responses. For instance, viral antigen-transfected fibroblasts induce Class I restricted CTL in vivo in mice but fail to do so in vitro (Kundig, et al., Science 268:1343-1347 (1995)). Therefore, an antibody induction study with GM-CSF fusion proteins in vivo does not imply any of its in vitro utilities, and is particularly unpredictive of CTL induction in vitro or in vivo.
Other methods that have been used for in vitro induction of primary protein-derived CTL are osmotic shock of dendritic cells and the use of Ph-sensitive liposomes (Nair, et al., J. Exp. Med. 175:609-12 (1992)). However, such methods have been shown to be inherently ineffective and toxic to APC's, because they disrupt cellular membranes by physical and chemical force in order to release the protein antigen into the cytoplasm.
These limitations are overcome by the discovery encompassed by the present invention. It is the discovery of the present invention that a T-cell response, and specifically, an MHC-Class I mediated T-cell response, can be stimulated by an isolated or soluble protein, when it is presented to the immune system as part of a complex with a dendritic cell binding protein, and more particularly GM-CSF. It is the further discovery of the present invention that such a response can be stimulated in vitro. As discussed above, in vitro stimulation of such a response has not previously been demonstrated using a full-length soluble antigen. The present invention provides for induction by isolated or soluble proteins of cellular immunity in vitro by presenting a specific antigen to an antigen presenting cell (APC), such as a dendritic cell, as part of an immunogenic fusion protein.
An important aspect of the present invention is the choice of fusion partner protein, a dendritic cell binding protein, such as granulocyte-macrophage colony stimulating protein (GM-CSF). Without relying on any particular mechanistic theory, it is believed that the protein antigen is transported over the plasma membrane of the APC in a receptor mediated non-disruptive way. It is further believed that the dendritic cell binding portion of the fusion protein serves to preserve the viability and functionality of the APC.
An additional aspect of the invention relates to the choice of target antigen. Although several tumor related antigens have been shown to serve as targets for T-cell mediated immunity in vivo, in vitro induction by isolated soluble polypeptide antigens has not been demonstrated. (Fisk, et al., J. Exp. Med. 181:2109-2117 (1995)). In experiments carried out in support of the present invention, it has now been demonstrated that tumor associated proteins not been previously shown to be a target antigens for CTL can become such targets by priming CTL with GM-CSF fusion derivatives in vitro.